microRNA-193a-5p Suppresses the Migratory Ability of Human KATO III Gastric Cancer Cells through Inhibition of Vimentin and MMP-9.

Purpose: microRNA-193a-5p is one of the well-known tumor suppressor miRNAs in the body but in many cases, its expression became reduced in patients suffering from gastric cancer (GC). The main purpose of this study was to restore the function of this miRNA in human GC cells and investigating the effects of enhanced expression of miR-193a-5p on proliferation, apoptosis, and migration of GC cells upon in vitro transfection. Methods: The KATO III GC cells were treated with 100 nM of miR-193a-5p or negative control sequences. Following that, the MTT assay, flow cytometry assay, and wound-healing assay were applied to estimate the impacts of enhanced expression of this miRNA on the viability, apoptosis, and migration rate of the cells, respectively. Moreover, the total RNA was isolated and alterations in the mRNA expression ratio of migratory genes were measured by qRT-PCR techniques. Results: The findings designated that enhanced expression of miR-193a-5p suppressed the migratory ability of the cells, but had no significant effects on cell survival or apoptosis of the transfected cells. In addition, this inhibitory function of miR-193a-5p on the migration rate of the KATO III cell line occurs with concurrent suppression of vimentin and MMP-9 gene expression. Conclusion: It can be concluded that miR-193a-5p negatively influences the migratory ability of the cancerous cells and restoring its effects can be regarded as a promising target of future therapeutic interventions, especially for GC metastasis.

Evaluation of Cellular Immune Responses in Dogs Immunized with Alum-Precipitated Autoclaved Leishmania major along with BCG and Imiquimod.

BACKGROUND: We aimed to investigate the potential effects of BCG and imiquimod on improvement of current experimental L. major vaccine against dogs in an endemic area of Zoonotic visceral leishmaniasis (ZVL) in Iran. METHODS: During 2012 till 2014, seven mixed-breed shepherd dogs with no anti-Leishmania antibodies and no response to Leishmanin reagent were immunized with 2 doses of alum-precipitated autoclaved L. major (Alum-AML) while BCG and imiquimod (for skin pre-treatment) were used as adjuvants. The productions of a few characteristic cytokines of T-helper immune responses and the development of delayed-type hypersensitivity (DTH) of the immunized animals were then evaluated, up to 300 days. Blood samples were collected at 0, 30, 80 and 300 d post-vaccination and the concentrations of IFN-γ, IL10, IL-12 and TGF-ß cytokines secreted from PBMCs at these time-points were quantified by ELISA. DTH was evaluated by Leishmanin skin test (LST). RESULTS: Although a similar LST conversion was observed at all time-points, the cytokine measurement results indicated significantly higher levels of IFN-γ at day 80 and elevated levels of IL-10 at days 80 and 300, post-vaccination. Moreover, a significantly higher IFN-γ/IL-10 ratio was observed at day 30 post-vaccination compared to the other time-points. CONCLUSION: Although a Th1-like response could be observed at day 30 post-vaccination, the development of cytokine profiles was inclined toward mixed Th1 and Th2 responses at days 80 and 300 post-vaccination. This situation may indicate the requirement of an additional boosting by this Alum-AML formula, in order to induce long-lasting protection against ZVL.

Combination therapy with miR-34a and doxorubicin synergistically induced apoptosis in T-cell acute lymphoblastic leukemia cell line.

MicroRNAs are identified to take actively part in the development of different cancers. Reduced expression of tumor suppressor miRNAs leads to cancer cell development, so restoring the expression of these miRNAs can be an appropriate treatment option for cancer. Due to the heterogeneity of cancer cells, single-drug therapy often results in drug resistance. Therefore, the combination of chemotherapy with miRNA can be a powerful strategy for cancer treatment. In the current investigation, miR-34a mimic, and negative control were purchased and transfected using jetPEI reagents. Then the synergic effects of miR-34a in combination with doxorubicin were investigated on cell death of acute T-cell lymphoblastic leukemia Jurkat cell line, as well as the expression of some genes including Caspase-3, Bcl-2, and p53 which are involved in apoptosis. Our outcomes showed that this combination remarkably reduced the expression of the Bcl-2 gene, the target gene of miR-34a. According to the results of the MTT assay, the survival rate was significantly decreased compared to the untreated cells. Results of the flow cytometry assay and DAPI staining demonstrated an increased apoptosis rate of Jurkat cells in combination therapy. Moreover, cell cycle arrest was observed at the G2/M phase in cells that were treated with miR-34a/doxorubicin. Most importantly, we showed that the transfection of the Jurkat cells with miR-34a increased the sensitivity of these cells to doxorubicin. Furthermore, the combination of miR-34a and doxorubicin drug effectively increased apoptosis of treated cells. Therefore, this method can be used as an impressive treatment for T-ALL.

The Impact of Nrf2 Silencing on Nrf2-PD-L1 Axis to Overcome Oxaliplatin Resistance and Migration in Colon Cancer Cells.

BACKGROUND: Nuclear factor-erythroid 2-related factor 2 (Nrf2) plays a key role in promoting chemoresistance in various cancers. PD-L1 is one of the downstream targets of the Nrf2 signaling pathway. This molecule has some beneficial impacts on tumors growth by inhibition of the immune system. This study aimed to investigate the potential role of the Nrf2-PD-L1 axis in the promotion of oxaliplatin resistance in colon cancer cells. METHODS: We examined Nrf2, PD- L1, and CD80 expression in the tumor and margin tissue samples from CRC patients. After that role of the Nrf2-PD-L1 axis in promotion of Oxaliplatin resistance was investigated. RESULTS: Our data revealed that Nrf2 and PD-L1 mRNA expressions were markedly higher in tumor tissues compared to margin tissues. The PD-L1 mRNA expression level was also increased in the resistant cells. However, Nrf2 expression was decreased in SW480/Res cells and increased in LS174T/Res cells. The inhibition of Nrf2 through siRNA treatment in SW480/Res and LS174T/Res cells has decreased the IC50 values of oxaliplatin. Inhibition of the Nrf2 has significantly increased the oxaliplatin-induced apoptosis, and reduced the migration in SW480/Res cells. CONCLUSION: It is suggested that effective inhibition of Nrf2-PD-L1 signaling pathways can be considered as a novel approach to improve oxaliplatin efficacy in colon cancer patients.

miR-34a and miR-200c Have an Additive Tumor-Suppressive Effect on Breast Cancer Cells and Patient Prognosis.

Breast cancer is the most common women's malignancy in the world and, for subgroups of patients, treatment outcomes remain poor. Thus, more effective therapeutic strategies are urgently needed. MicroRNAs (miRNAs) have emerged as promising therapeutic tools and targets, as they play significant roles in regulating key cellular processes by suppressing gene expression. However, additive opportunities involving miRNAs have been underexplored. For example, both miR-34a and miR-200c individually suppress the development of different types of cancer, but the cellular effects of their combined actions remain unknown. Here, we show that miR-34a and miR-200c levels are reduced in breast tumors compared to adjacent normal tissues and that this additively predicts poor patient survival. In addition, in cell lines, miR-34a and miR-200c additively induce apoptosis and cell cycle arrest, while also inhibiting proliferation, invasion, migration, stemness and epithelial-to-mesenchymal transition (EMT). Mechanistically, both miRNA-34a and miR-200c directly target HIF1-α and subsequently downregulate VEGFR, MMP9 and CXCR4, although combined miRNA-34a and miR-200c delivery suppresses mouse xenograft tumor development as effectively as individual delivery. We establish a model, supported by in vitro and clinical data, which collectively suggest that the co-delivery of miR-34a and miR-200c represents a promising novel therapeutic strategy for breast cancer patients.

HMGA2 as a Critical Regulator in Cancer Development.

The high mobility group protein 2 (HMGA2) regulates gene expression by binding to AT-rich regions of DNA. Akin to other DNA architectural proteins, HMGA2 is highly expressed in embryonic stem cells during embryogenesis, while its expression is more limited at later stages of development and in adulthood. Importantly, HMGA2 is re-expressed in nearly all human malignancies, where it promotes tumorigenesis by multiple mechanisms. HMGA2 increases cancer cell proliferation by promoting cell cycle entry and inhibition of apoptosis. In addition, HMGA2 influences different DNA repair mechanisms and promotes epithelial-to-mesenchymal transition by activating signaling via the MAPK/ERK, TGFß/Smad, PI3K/AKT/mTOR, NFkB, and STAT3 pathways. Moreover, HMGA2 supports a cancer stem cell phenotype and renders cancer cells resistant to chemotherapeutic agents. In this review, we discuss these oncogenic roles of HMGA2 in different types of cancers and propose that HMGA2 may be used for cancer diagnostic, prognostic, and therapeutic purposes.

STAT3 Silencing and TLR7/8 Pathway Activation Repolarize and Suppress Myeloid-Derived Suppressor Cells From Breast Cancer Patients.

Cancer cells escape immune destruction. From this perspective, myeloid-derived suppressor cells (MDSCs), which are immunosuppressive in various cancers including breast cancer (BC), are significant. However, the precise mechanisms are unknown. We isolated HLA-DR-CD33+ MDSCs and CD3+ T cells from BC patients' peripheral blood and healthy donors through MACS and immunophenotyped by flow cytometry. Transfection of short-interfering RNAs and treatment with a TLR7/8 agonist altered pathway activities in vitro. Gene expression was analyzed using qRT-PCR, western blotting, and immunohistochemistry. Our findings showed an association between the progression of BC and increased levels of circulating HLA-DR-CD33+ MDSCs. These cells strongly suppress both autologous and analogous CD3+ T cell proliferation and enter the tumor microenvironment. We also identified increased STAT3 signaling and increased IDO and IL-10 expression in BC-derived MDSCs as immunosuppression mechanisms. Further, STAT3 inhibition and TLR7/8 pathway stimulation reduce the immunosuppressive activity of patient-derived MDSCs on T cells by inducing MDSC repolarization and differentiation into mature myeloid cells. This also alters the expression of critical cytokines and transcription factors in CD3+ T cells and, importantly, reduces breast cancer cells' proliferation. Finally, while chemotherapy is able to significantly reduce circulating MDSCs' level in patients with breast cancer, these MDSCs remained highly T cell-suppressive. We identified a novel molecular mechanism of MDSC-mediated immunosuppression. STAT3 inhibition and TLR7/8 pathway stimulation in MDSCs repolarize and suppress MDSCs from breast cancer patients. This offers new opportunities for BC immunotherapy.

miR-330 suppresses EMT and induces apoptosis by downregulating HMGA2 in human colorectal cancer.

MicroRNAs (miRNAs) are important molecular regulatorsof cellular signaling and behavior. They alter gene expression by targeting messenger RNAs, including those encoding transcriptional regulators, such as HMGA2. While HMGA2 is oncogenic in various tumors, miRNAs may be oncogenic or tumor suppressive. Here, we investigate the expression of HMGA2 and the miRNA miR-330 in a patient with colorectal cancer (CRC) samples and their effects on oncogenic cellular phenotypes. We found that HMGA2 expression is increased and miR-330 expression is decreased in CRCs and each predicts poor long-term patient survival. Stably increased miR-330 expression in human colorectal cancer cells (HCT116) and SW480 CRC cell lines downregulate the oncogenic expression of HMGA2, a predicted miR-330 target. Additionally, this promotes apoptosis and decreases cell migration and viability. Consistently, it also decreases protein-level expression of markers for epithelial-to-mesenchymal-transition (Snail-1, E-cadherin, and Vascular endothelial growth factor receptors) and transforming growth factor ß signaling (SMAD3), as well as phospho- Protein kinase B (AKT) and phospho-STAT3 levels. We conclude that miR-330 acts as a tumor suppressor miRNA in CRC by suppressing HMGA2 expression and reducing cell survival, proliferation, and migration. Thus, we identify miR-330 as a promising candidate for miRNA replacement therapy for patients with CRC.

Effects of CD133 Silencing on Survival and Migration of HT-29 Colorectal Cancer Cells.

BACKGROUND: Colorectal cancer (CRC) is attributed as one of the most common malignancies worldwide. CD133 molecule, as a pentaspan transmembrane glycoprotein, confers stem cell-related characteristics, including self-renewal and multi-directional differentiation capability. CD133 plays important roles in the progression of CRC by conferring apoptotic resistance and migration ability. OBJECTIVE: To investigate the anti-apoptotic and anti-angiogenic effect of CD-133 targeted siRNA in a colorectal cancer cell line. METHODS: In this study, CD133-targeted siRNA transfection was conducted into HT-29 cells. MTT assay was employed to evaluate the cytotoxic effects of transfection on the cells. Flow cytometry was used to evaluate the apoptosis rate. The mRNA expression of apoptosis and metastasis related genes were assessed by quantitative Real-Time PCR (qRT-PCR). Wound healing assay was used to assess the migration potency of the infected cells. RESULTS: Expression of CD133 was significantly downregulated after transfection of CD133-specific siRNA. Moreover, the rate of apoptosis was significantly increased after transfection. The migration potential of cells was diminished after transfection. siRNA delivery resulted in the modulation of expression of apoptosis and metastasis-related genes. CONCLUSION: siRNA mediated targeting of CD133 could be considered as a promising approach to treat CRC through suppressing the cancerous behavior of tumor cells.

Photodynamic therapy using zinc phthalocyanine with low dose of diode laser combined with doxorubicin is a synergistic combination therapy for human SK-MEL-3 melanoma cells.

Chemotherapy is a generally used anticancer strategy for melanoma and it may have improved outcomes in combination with other approaches. One such strategy is photodynamic therapy (PDT), where a photosensitizer (PS) generates reactive oxygen species (ROS) after illumination of target cells. Interestingly, in low doses and high doses of light sources, special cellular responses can be induced. Regarding this fact, in this study, the combination of zinc phthalocyanine (ZnPc)-PDT and Doxorubicin (DOX) was applied at low and high dose of diode laser to treat SK-MEL-3 cells. Cytotoxic effects were determined by MTT assay for assessment synergistic effects were estimated by calculation of Combination Index (CI); that synergistic effects were observed in most groups. In low dose of laser irradiation higher synergism effects were observed. Significant changes of ROS were not observed with combinations, but autophagy, subG1 and G2/M phase cell cycle arrest, decreased cell migration ability and apoptosis induction were significantly increased compared to either treatment alone. The expression of caspase-8, -9, -3 and Bcl-2 genes revealed caspase-dependent apoptosis in all groups. Moreover, ZnPc-PDT and chemo-PDT down-regulated the expression of MMP-9 and Vimentin genes that impaired cell migration. In conclusion, it can be suggested that pre-treatment with ZnPc-PDT has high effects to sensitize SK-MEL-3 cells to DOX, in particular with low dose of diode laser.

miR-142-3p is a tumor suppressor that inhibits estrogen receptor expression in ER-positive breast cancer.

Estrogen receptors (ERs) are involved in the development of many types of malignant tumors, in particular, breast cancer. Among others, ERs affect cell growth, proliferation, and differentiation. The microRNA (miRNA) miR-142-3p has been shown to inhibit carcinogenesis by regulating various cellular processes, including cell cycle progression, cell migration, apoptosis, and invasion. It does so via targeting molecules involved in a range of signaling pathways. We surgically collected 20 ER-positive breast cancer samples, each with matched adjacent normal breast tissue, and measured the expression of miR-142-3p via quantitative real-time polymerase chain reaction (qRT-PCR). Bioinformatics methods, luciferase reporter assay, qRT-PCR, and western blot analysis were used to assess whether miR-142-3p could target ESR1, which encodes the estrogen receptor, in ER-positive breast cancer cells and patient samples. We also restored miRNA expression and performed cell viability, cytotoxicity, and colony formation assays. Western blot analysis and qRT-PCR were used to study the expression of apoptosis and stemness markers. We found that miR-142-3p is downregulated in ER-positive breast cancers. Restoration of miR-142-3p expression in ER-positive breast cancer cells reduced cell viability, induced apoptosis via the intrinsic pathway and decreased both colony formation and the expression of stem cell markers. Bioinformatic analysis predicted miR-142-3p could bind to 3'-untranslated region ESR1 messenger RNA (mRNA). Consistently, we demonstrated that miR-142-3p reduced luciferase activity in ER-positive breast cancer cells, and decreased ESR1 expression in both mRNA and protein levels. The results revealed miR-142-3p and ESR1 expression correlated negatively in ER-positive breast cancer samples. The results suggest miR-142-3p acts as a tumor suppressor via multiple mechanisms. Thus, restoration of miR-142-3p expression, for example, via miRNA replacement therapy, may represent an effective strategy for the treatment of ER-positive breast cancer patients.

Transcript Level of MicroRNA Processing Elements in Gastric Cancer.

BACKGROUND: Aberrant expression of microRNAs (miRNAs) has been implicated in the etiopathogenesis and development of various cancers. Drosha and Dicer are the main components of the miRNA biosynthesis machine. Another enzyme, DGCR8, is the assistant of Drosha in the processing complex. Here, we tried to evaluate the mRNA transcript level of Drosha, Dicer, and DGCR8 genes in involved tissues from patients with gastric cancer. METHODS: Fifty tumoral and their marginal tissues, as the control group, were obtained from patients with gastric cancer. After RNA extraction from tissues and cDNA synthesis, quantification of mRNA expression of Drosha, Dicer, and DGCR8 was conducted using SYBR Green master mix and real-time PCR. RESULTS: It was observed that mRNA expression levels of Drosha, Dicer, and DGCR8 were significantly upregulated in tumoral tissues compared with marginal tissues. Upregulation of these genes was not correlated with clinical manifestations of the patients. CONCLUSIONS: Upregulation of Drosha, Dicer, and DGCR8 plays a role in the development of cancer, probably through dysregulated the expression level of miRNAs.

Circulating myeloid-derived suppressor cells: An independent prognostic factor in patients with breast cancer.

Evading immune destruction is a hallmark of cancer. Myeloid-derived suppressor cells (MDSCs), a heterogeneous population of myeloid immune cells, are thought to foster the establishment of an immunosuppressive tumor microenvironment, but it remains unclear how. This study aims to determine the levels of circulating MDSCs and their subpopulations and test their immunosuppressive functions in patients with breast cancer (BC). We analyzed the fractions of MDSCs in freshly isolated peripheral blood mononuclear cells of patients with BC and healthy donors using flow cytometry. Circulating MDSCs were further phenotyped using fluorescently labeled antihuman monoclonal antibodies. Coculture experiments revealed the effects of MDSCs on CD3+ T cell response. Moreover, we correlated circulating MDSC levels with clinicopathological features of patients with BC. We show that the fraction of HLA-DR - CD33 + MDSCs in peripheral blood is about 10-fold higher in patients with BC than in healthy control individuals. The levels of all MDSC subpopulations, including monocytic and granulocytic MDSCs, are significantly elevated. Coculture experiments of purified HLA-DR - CD33 + MDSCs and CD3 + T cells demonstrate that T cell proliferation is more effectively inhibited by BC patient-derived MDSCs than by healthy control MDSCs. Moreover, increased circulating MDSC levels robustly associate with advanced BC stage and positive lymph node status. By being more abundant and more effective T cell suppressors, BC patient-derived circulating MDSCs exert a dual immunosuppressive effect. Our findings pave the way to develop novel diagnostic and immunotherapeutic strategies, aimed at detecting and inhibiting MDSCs in patients with BC.

Let-7a Could Serve as A Biomarker for Chemo-Responsiveness to Docetaxel in Gastric Cancer.

BACKGROUND: MicroRNAs are noncoding RNAs which play critical roles in response to anti-cancer agents. Let-7a and miR-21 are well-known tumor-suppressor and oncomiR miRNAs, respectively. They are involved in tumorigenesis of gastric cancer and have potential to be used as markers in response to the therapy. OBJECTIVE: We aimed to study alterations in the expression of Let-7a and miR-21, and their targets in gastric cancer cell lines after treatment with docetaxel. METHODS: In order to determine the IC50 of docetaxel, MTT assay was performed in AGS, MKN45 and KATO III gastric cancer cell lines. The expression levels of Let-7a and miR-21 and their target genes, HMGA2 and PDCD4, were determined by reverse-transcription quantitative real-time PCR for both treated and untreated cell lines. RESULTS: MTT assay showed higher IC50 concentration of docetaxel in KATO III in comparison with AGS and MKN45, indicating KATO III`s higher resistance to docetaxel. Following the treatment, the expression level of Let-7a was significantly increased in AGS and MKN45, while decreased in KATO III. Expression level of miR- 21 in the three treated cell lines was increased significantly. Not only Let-7a, but also expression level of HMGA2 and PDCD4 genes showed different patterns in KATO III in comparison with AGS and MKN45. CONCLUSION: Down-regulation and up-regulation of Let-7a in docetaxel-resistant and sensitive cell lines, respectively indicates its potential usefulness as biomarker for responsiveness of gastric cancer to the therapy with docetaxel and also for predicting patient`s outcome.

microRNA-193a-5p inhibits migration of human HT-29 colon cancer cells via suppression of metastasis pathway.

PURPOSE: Altered expression of microRNAs (miRNAs) is indicated strongly in colorectal cancer (CRC). This study aims to evaluate the inhibitory role of miR-193a-5p on epithelial-mesenchymal transition markers in CRC lines. The cellular effects and potential mechanisms of miR-193a-5p were also examined. METHODS: Quantitative reverse-transcription polymerase chain reaction (RT-PCR) was performed to determine the expression of miR-193a-5p in three CRC cell lines (HCT-116, SW-480, and HT-29) and its impact on metastasis-related genes ( vimentin and CXCR4) before and after mimic transfection. Of those, the cell line with the highest changes was selected for the next upcoming experiments such as wound-healing assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and annexin-V staining tests. RESULTS: Our results revealed that miR-193a-5p was significantly downregulated in three CRC cell lines and that HT-29 displayed the most decrease ( P < 0.0001). The restoration of miR-193a-5p in human HT-29 cell line inhibited cell migration. But, miR-193a-5p transfection did not affect cell viability and had no significant effect on apoptosis induction. Also, the quantitative RT-PCR analysis of miR-193a-5p mimic transfected cells revealed a significant increase in miR-193a-5p messenger RNA (mRNA) expression level ( P < 0.0001) with reduction of vimentin and CXCR4 mRNA expression levels in HT-29 cell line ( P < 0.01 and < 0.05, respectively). CONCLUSION: Our results indicated that miR-193a-5p acts as a tumor suppressor miRNA and its downregulation plays an important role in metastasis via upregulation of metastasis-related genes in CRC. Therefore, it can be considered as a potential therapeutic target for applying in CRC management in the future.

Targeting of high mobility group A2 by small interfering RNA-loaded nanoliposome-induced apoptosis and migration inhibition in gastrointestinal cancer cells.

BACKGROUND: Considering the complex nature of gastrointestinal cancer, different methods including surgery, radiotherapy, and chemotherapy are considered for the treatment. Novel strategies including silencing of oncogenes using safe delivery systems could be considered as a novel approach in colorectal cancer treatment. The aim of this study was to investigate the silencing effect of high mobility group A2 (HMGA2) small interfering RNA (siRNA)-loaded nanoliposomes on gastrointestinal cancers. METHODS: The siRNA-lipoplexes were prepared using dioleoyl trimethylammonium propane (DOTAP)/cholesterol (Chol)/1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) through the freeze-drying of a monophase solution method. The size, polydispersity index (PDI), and zeta-potential of nanoliposomes were determined using Zetasizer analyzer. The morphology of the nanoliposomes was determined by transmission electron microscopy (TEM). The agarose gel-retardation assay was carried out to confirm the loading of siRNAs into liposome. The silencing of the HMGA2 in cancer cells was evaluated by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The effect of liposomes on cell cytotoxicity was studied by MTT assay. The inhibitory effect of siRNA-loaded liposomes was evaluated by a wound-healing assay. The apoptosis induction was investigated via the annexin V/propidium iodide assay. RESULTS: The size, PDI, and zeta-potential of the prepared liposomes were found to be 350 nm, 0.67, and 86.3 mV, respectively. They were spherical in shape and could efficiently associate with siRNA. The results of gene silencing showed that the optimum condition of HMGA2 silencing was 80 pmol HMGA2 and 24 hours after treatment in each cancer cell lines. MTT assays indicated that silencing of HMGA2 in optimal condition could reduce the viability of the cancer cells more than 60% in the three cell lines. The result of the apoptosis assay showed more than 50% of the cell deaths related to the apoptosis in all three cell lines. The gene expression evaluation confirmed that apoptosis was induced via the intrinsic pathway inducing both caspase-3 and -9 expressions. Also, the reduction in Bcl2 expression confirmed the activation apoptosis pathway in the treated cancer cells. The wound-healing assay showed the suppression of cancer cell migration after treatment with the prepared nanoliposomes. CONCLUSION: The results of this study showed the HMGA2 siRNA-loaded nanoliposomes could be effective in the treatment of gastrointestinal cancers.

MicroRNA-145 replacement effect on growth and migration inhibition in lung cancer cell line.

BACKGROUND: Lung cancer is the main cause of cancer death in males and females worldwide. Reduced expression of miR-145 has been reported in many types of cancers. In this study, we transfected miR-145 into lung cancer cells by vector-based miR-145, and investigated the effects of this intervention on growth and migration inhibition of cancer cells as well on the expression of targeted genes. METHODS: IC50 of Geneticin (G418) antibiotic was measured using MTT test in NSCLC cell lines. miR-145 was transfected into lung cancer cells by jetPEI. qRT-PCR was used to evaluate the transcript level of the miR-145 and expression for KRAS, MMP-9, vimentin, caspase-3, caspase-8 and caspase-9 genes in A549 cells. MTT assay was used to evaluate the proliferation inhibition of cancer cells. Wound healing assay was used to check the migration status of transfected lung cancer cells. The apoptosis induction was assessed by DAPI staining assay. RESULTS: The MTT assay showed that the IC50 of Genticin was 494.1 µg/ml. The results of the qRT-PCR showed increased expression level of miR-145 and downregulation of KRAS, MMP-9, and vimentin expression in A549 transfected cells compared with the control group. The MTT assay results demonstrated inhibition of cancer cell proliferation after miR-145 replacement. Wound healing assay results revealed that migration was reduced upon miR-145 transfection. The transfected cell displayed increased apoptosis rate by inducing caspase-3 and caspase-9 mRNA expression. CONCLUSION: The results of this study showed that increased miR-145 expression exerted a critical role in subsiding the growth, survival, and migration of lung cancer cell line.

PTPN22 Silencing in Human Acute T-Cell Leukemia Cell Line (Jurkat Cell) and its Effect on the Expression of miR-181a and miR-181b.

Purpose: T-cell acute lymphoblastic leukemia (T-ALL) is one of the most common malignancies associated with T-lymphocytes, accounting for 10 to 15 percent of ALL cases in children and 25 percent in adults. Innovative therapeutic approaches that overcome ineffective treatments on tumor cells may be a potential source of improvement in therapeutic approaches. Suppression of gene expression at transfusion level is one of the important strategies in gene therapy. The expression of PTPN22 and miR-181 genes in all types of hematologic malignancies increases and is likely to contribute to the survival and death of cells by affecting a variety of signaling pathways. The purpose of this study was to determine the role of PTPN22 inhibition by siRNA, and alteration in miR-181a and miR-181b in Jurkat cell line. Methods: Jurkat cells were transfected with 80 pmol of siRNA to inhibit PTPN22. After that, expression of PTPN22 mRNA and transcript levels of miR-181a and miR-181b were measured with Real-time PCR after 48hrs. Results: Experiments demonstrated that siRNA transfection resulted in significant downregulation of PTPN22 mRNA after 48 hrs in 80 pmol dose of siRNA. Moreover, transcript levels of both miR-181a and miR-181b was decreased after transfection. Conclusion: PTPN22, miR-181a and miR-181b might be involved in progression of Jurkat cells and targeting these molecules by RNAi might confer promising tool in treatment of T-ALL.

Differential altered expression of let-7a and miR-205 tumor-suppressor miRNAs in different subtypes of breast cancer under treatment with Taxol.

BACKGROUND: MicroRNAs (miRNAs) are small non-coding RNAs which have been considered as major players in the process of carcinogenesis and drug responsiveness of breast cancer. OBJECTIVES: In this study, we aimed to investigate the expression pattern of let-7a and miR-205 tumorsuppressor miRNAs in breast cancer cell lines under treatment with paclitaxel. MATERIAL AND METHODS: The half maximal inhibitory concentration (IC50) of paclitaxel was determined for 4 breast cancer cell lines, including MCF-7, MDA-MB-231, SKBR-3, and BT-474 by an MTT assay. The expression level of let-7a and miR-205, and their targets, K-RAS and HER3, was determined before and after treatment with paclitaxel, using quantitative reverse transcriptase real-time polymerase chain reaction (qRT-PCR). RESULTS: After treatment, the expression level of both let-7a and miR-205 was significantly increased in HER2- overexpressing cell line BT-474 (26.4- and 7.2-fold, respectively). In contrast, the HER2-negative cell lines MCF-7 and MDA-MB-231 showed a significantly decreased expression of both let-7a (30.3- and 13.5-fold, respectively) and miR-205 (20- and 18.1-fold, respectively). Controversially, SKBR-3 revealed a significantly decreased expression of both let-7a (1.3-fold) and miR-205 (1.3-fold). The expression level of K-RAS as a target of let-7a decreased in all cell lines significantly, but the pattern of alteration in the expression level of HER3 as a target of miR-205 in all cell lines was the reverse of the pattern of alteration in the expression level of miR-205. CONCLUSIONS: Our results confirmed a better response of HER2-overexpressing breast cancer to paclitaxel at the miRNA level. One putative reason could be the upregulation of tumor-suppressor miRNAs after treatment with paclitaxel. On the other hand, HER2-negative breast cancer cell lines showed a significantly decreased expression of tumor-suppressor miRNAs, a putative mechanism of resisting the therapy.

Nrf2/P-glycoprotein axis is associated with clinicopathological characteristics in colorectal cancer.

Colorectal cancer (CRC) is the fourth leading cause of cancer-related death worldwide. Activation of ABCB1 gene and its main product, P-glycoprotein, is the common reason for chemoresistance. The nuclear factor-erythroid 2-related factor2 (Nrf2) is directly regulated by Kelch like ECH-associated protein1 (Keap1). In addition, Nrf2 is a key transcriptional factor that regulates efflux transporters, including P-gp. The aim of this study was to investigate the expression levels of Nrf2, Keap1 and ABCB1 in the biopsy samples and their association with clinicopathological features in CRC patients. Both mRNA and protein expression levels were measured by Real-time PCR and immunohistochemistry (IHC), respectively, in biopsies from colonoscopy in 65 CRC patients compared to those in 65 non-CRC individuals. While expression levels of Nrf2 and ABCB1 (P-gp) were markedly higher in both mRNA and protein levels in CRC biopsies (p < 0.01), Keap1 expression level was significantly lower in these samples (p < 0.05). Positive correlations between Nrf2 expression level and tumor size (p = 0.003), lymph node (p = 0.038), distant metastasis (p = 0.008), and smoking status (p = 0.02) were observed. However, P-gp expression was associated only with patient age and smoking status. In addition, there was a positive correlation between protein levels of Nrf2 and P-gp, in both CRC (r = 0.617, p < 0.001) and non-CRC tissues (r = 0.930, p < 0.001). In conclusion, over-expression of Nrf2 and ABCB1/P-gp, as well as down-regulation of mRNA expression level of Keap1 in CRC patients denotes the role of Keap1/Nrf2/ABCB1 axis in CRC progression and chemoresistance. Our data suggest that therapeutic inhibition of Nrf2/ABCB1 signaling can be considered as a novel strategy to improve the efficacy of chemotherapeutics against CRC.